epr of transition metal ionsbh.knu.ac.kr/~leehi/index.files/epr_for_bioinorganic... ·...

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EPREPR of Transition Metal Ionsof Transition Metal IonsApplications of EPR for Bioinorganic Researches

이홍인

경북대학교 생무기화학실험실(053) 950-5904

[email protected]://bh.knu.ac.kr/~leehi

"But don't you see what this implies? It means that there is a fourth degree of freedom for the electron. It means that the electron has spin, that it rotates."

- George Uhlenbeck to Samuel Goudsmit in 1925 on hearing of the Pauli principle -

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Zavoisky가 1944년에 얻은 최초의 EPR 스펙트럼(시료: CuCl2.2H2O, 자장의 세기: 47.6G, 전자기파의 주파수: 133MHz)

ApplicationsApplications

Anthropology, Archeology, Biochemistry, Biology, Chemical Reactions, Clusters, Colloids, Coal, Dating, Dosimetry, Electrochemistry, EPR Imaging, Excitons, Ferromagnets, Forensic Science, Gases, Gemmology, Geography, Geology, Glass, History, Inorganic Radicals, Materials Science, Medicine, Metal Atom Chemistry, Metalloproteins, Microscopy, Mineralogy, Organic

Radicals, Organometallic Radicals, Paleontology, Photochemistry, Photosynthesis, Point Defects, Polymers, Preservation Science, Quantum Mechanics, Radiation Damage, Semiconductors, Spin Labels, Spin Traps,

Transition Metals, Zoology

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EPR MethodologiesEPR Methodologies

EPR

CW-EPR Pulsed-EPR

EPR

ENDOR

FID Spin Echo

ENDOR ESEEMFT-EPR

Mims ENDOR

Davis ENDOR

2,3,4-pulse

HYSCORE

ELDOR

ELDOR

Imaging

DEER

2+1

These are just scratches of modern EPR techniques.

What is EPR ?What is EPR ?Electron Pramagnetic Resonance (EPR)Electron Spin Resonance (ESR)Electron Magnetic Resonace (EMR)

“Electron Zeeman Interaction”

N S

S N

higher energy state (mS = ½)

lower energy state (mS = -1/2)

N S

EPR ~ ESR ~ EMR

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What is EPR ?What is EPR ?


N S

S N

higher energy state (mS = ½)

lower energy state (mS = -1/2)

N S

hν (microwave)

EPR is the resonant absorption of microwave radiationby paramagnetic systems in the presence of an applied magnetic field.



Conventional CW EPR spectrometer Arrangement

S = 1/2

ms = + 1/2

ms = - 1/2

hν (= gβBo)

Bo

h Planck’s constant (6.626196 x 10-27 erg.sec)ν frequency (GHz or MHz)g g-factor (approximately 2.0)β Bohr magneton (9.2741 x 10-21 erg.Gauss-1)Bo magnetic field (Gauss or mT)

Selection Rule∆MS = ±1

H = βS.g.H

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Bruker EMX EPR spectrometer

S = 1/2

ms = + 1/2

ms = - 1/2

hν (= gβBo)

Bo



H = βS.g.H



경북대학교

S = 1/2

ms = + 1/2

ms = - 1/2

hν (= gβBo)

Bo



H = βS.g.H

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Magnetic Field (Bo)

∆Bpp


g = hν/βBo

S = 1/2

ms = + 1/2

ms = - 1/2

hν (= gβBo)

Bo



H = βS.g.H

H = βS.g.H

What is What is gg ??

Magnetic Field (Bo)

∆Bpp

g = hν/βBo

It is an inherent property of a system containing an unpaired spin.

Similar to the chemical shift observed in an NMR spectrum.

The g value for a single unpaired electron (free electron) has been calculated and experimentally determined. It is 2.0023192778 ± 0.0000000062 (=ge). The g value for an S = 1/2 system is usually near ge, but it is not exactly at ge. Why not?

This is due to spin orbit coupling which determines both the value of g and its anisotropy (how far the 3 g values are from gav. The g value can often be calculated and the value is characteristic for a particular spin system. O O π-radical

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This is due to spin orbit coupling which determines both the value of g and its anisotropy (how far the 3 g values are from gav. The g value can often be calculated and the value is characteristic for a particular spin system.


N

NN

N

Ni

[Ni(cyclam)]3+

H

HH

H

Ni(III)d7

N

NN

N

Ni

tct-Ni(OEiBC)-

Ni(I)d9

O O π-radical


N

NN

N

Ni

[Ni(cyclam)]3+

H

HH

H

Ni(III)d7

N

NN

N

Ni

tct-Ni(OEiBC)-

Ni(I)d9

MO Scheme for Low-Spin d7,9

Complexes

for d9,gz = ge [1 + 8 λ / ∆E1] = g║

gx,y = ge [1 + 2λ / ∆E2] = g┴

λ: spin-orbit coupling constant

∆E1

∆E2

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N

NN

N

Ni

[Ni(cyclam)]3+

H

HH

H

Ni(III)d7


N

NN

N

Ni

tct-Ni(OEiBC)-

Ni(I)d9

MO Scheme for Low-Spin d7,9

Complexes

for d7,gz ~ ge = g║

gx,y = ge [1 + 6λ / ∆E1] = g┴

λ: spin-orbit coupling constant

∆E1

Powder Patterns of EPR SpectraPowder Patterns of EPR Spectragz

gx

gy

gx

Bo

Θ

Φ

Isotropic g: gx = gy = gz

Axial g: gx = gy ≠ gz

Rhombic g: gx ≠ gy ≠ gz

Even though we talk about gx, gy, and gz,the values should be more properly called g1, g2, and g3 unless we have evidence for the nature of the g tensor relative to themolecular axes.

Powder: randomly oriented samples such as frozen solutions, powders

geff = (gx2sin2θcos2φ+gy

2sin2θsin2φ+gz2cos2θ)1/2

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Powder Patterns of EPR SpectraPowder Patterns of EPR Spectragz

gx

gy

gx

Bo

Θ

Φ

geff = (gx2sin2θcos2φ+gy

2sin2θsin2φ+gz2cos2θ)1/2

Powder Patterns of EPR SpectraPowder Patterns of EPR Spectra

N

NN

N

Ni

[Ni(cyclam)]3+

H

HH

H

Ni(III)d7

N

NN

N

Ni

tct-Ni(OEiBC)-

Ni(I)d9

O O π-radical

Axial : gx = gy ≠ gz Rhombic: gx ≠ gy ≠ gz

Near isotropic

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N

NN

N

Ni

[Ni(cyclam)]3+

H

HH

H

Ni(III)d7

N

NN

N

Ni

tct-Ni(OEiBC)-

Ni(I)d9

EPR of EPR of Ni(I) Ni(I) and and Ni(III)Ni(III)

N

NN

N

Ni

[Ni(cyclam)]3+

H

HH

H

Ni(III)d7

N

NN

N

Ni

tct-Ni(OEiBC)-

Ni(I)d9

EPR of EPR of Cu(II) Cu(II) (S=1/2, d(S=1/2, d99))Cu(II)

?J. Braz. Chem. Soc. 17, 1501 (2006)

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This is due to spin orbit coupling which determines both the value of g and its anisotropy (how far the 3 g values are from gav. The g value can often be calculated and the value is characteristic for a particular spin system.

Powder EPRPowder EPR

N

NN

N

Ni

[Ni(cyclam)]3+

H

HH

H

Ni(III)d7

N

NN

N

Ni

tct-Ni(OEiBC)-

Ni(I)d9

O O π-radical

Solution EPRSolution EPR?

Powder: randomly oriented samples such as frozen solutions, powders

In solution: when molecules are rapidly tumbling (within microwave time scale), g-anisotropy is averaged out.

Electron spin Electron spin –– Nuclear spin InteractionNuclear spin Interaction

Beff = B0 - BInd Beff = B0 + BInd

“Hyperfine Interaction”nd Beff = B0 + BInd

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“Hyperfine Interaction”

S=½; I=½

Doublethfc (=A)

Selection Rule∆MS = ±1; ∆MI = 0

Magnetic Field

hfc: hyperfine coupling constant

S = 1/2

ms = + ½, mI = + ½

B0

A

hν (= ∆Ε)

ms = + ½, mI = - ½

ms = - ½, mI = - ½ms = - ½, mI = + ½

S = 1/2

ms = + ½, mI = + ½

B0

A

hν (= ∆Ε)

ms = + ½, mI = - ½

ms = - ½, mI = - ½ms = - ½, mI = + ½

H = βS.g.H + S.A.I

S = 1/2

ms = + ½, mI = + ½

B0

A

hν (= ∆Ε)

ms = + ½, mI = - ½

ms = - ½, mI = - ½ms = - ½, mI = + ½

S = 1/2

ms = + ½, mI = + ½

B0

A

hν (= ∆Ε)

ms = + ½, mI = - ½

ms = - ½, mI = - ½ms = - ½, mI = + ½

H = βS.g.H + S.A.I



S=½; I=½

Doublethfc (=A)

Selection Rule∆MS = ±1; ∆MI = 0

Magnetic Field

MS=±½

MS=-½

MS=+½

ElectronS(½)

MI=+½

MI=-½

MI=-½

MI=+½

NucleusI (½)

hfc: hyperfine coupling constant

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“Hyperfine Interaction”Selection Rule

∆MS = ±1; ∆MI = 0

MS=±½

MS=-½

MS=+½

ElectronS (½)

MS = ±½

MI=+1

MI=-1

MI=-1

MI=+1

NucleusI (1)

MI=0,±1

MI= 0

MI= 0 Magnetic Field

S=½; I=1

Triplet

hfc hfc

NO


“Hyperfine Interaction”Selection Rule

∆MS = ±1; ∆MI = 0

Magnetic Field

S=½; I=1/2 x 4Quintet

MS=±½

MS=-½

MS=+½

ElectronS(½)

MI=+4/2

4 NucleiI (½)

MI=+2/2

MI=0

MI=-2/2

MI=-4/2

MI=-4/2

MI=-2/2

MI=0

MI=+2/2

MI=+4/2

O O

H H

H H

hfc hfc hfc hfc

1 4 6 4 1

So far, we have considered the cases of hyperfine interactions in solutions or in the samples with very narrow g-anisotropy. How about powder samples?

Pascal’s triangle

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So far, we have considered the cases of hyperfine interactions in solutions or in the samples with very narrow g-anisotropy. How about powder samples?

For 61Ni, I = 3/2, so you expect (and see) 4 lines.

But the hyperfine splitting is unresolved in the g┴ direction. Note that the center of

the pattern is the g-value

Ni(I)d9


N

NN

N

Ni

[Ni(cyclam)]3+

H

HH

H

Ni(III)d7

14N (I=1)

Ni3+

N

HxO S-Cys

S-Cys

HN

N

N

Ni-SOD

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CuII d9

S=1/2; I=3/214N I=1

Superhyperfine Splitting

experiment

Sim axial S=1/2

Sim axial S=1/2A║ coupling,

due to I=3/2

Sim axial S=1/2A┴ and A║

coupling, due to I=3/2

EPR of EPR of Cu(II) Cu(II) (S=1/2, d(S=1/2, d99))

63Cu (69 %), I = 3/265Cu (31 %), I = 3/2

N

NN

N

Ni

[Ni(cyclam)]3+

H

HH

H

Ni(III)d7

N

NN

N

Ni

tct-Ni(OEiBC)-

Ni(I)d9

EPR of EPR of Co(II)Co(II) (S = 1/2, d(S = 1/2, d77))Co(II)59Co (100 %), I =7/2

FRBS Lett.. 409, 421 (1997)

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EPR of EPR of Co(II)Co(II) (S = 1/2, d(S = 1/2, d77))

FRBS Lett.. 409, 421 (1997)

Redox chemistry of cobalamin and iron-sulfur cofactors in the tetrachloroethene reductase of Dehalobacter restrictus

A : spectrum of 4mg/ml enzyme in 25mM Tris-HCl buffer pH 8.0 poised at a redox potetial of -27mV.

B : simulation of A

C : spectrum of enzyme in 125mM ches buffer, pH 9.6, poised at redox potential of -293mV.

D : C-A spectrum

E : simulation of D


FRBS Lett.. 409, 421 (1997)

Redox chemistry of cobalamin and iron-sulfur cofactors in the tetrachloroethene reductase of Dehalobacter restrictus

A : spectrum of 4mg/ml enzyme in 25mM Tris-HCl buffer pH 8.0 poised at a redox potetial of -27mV.

B : simulation of A

C : spectrum of enzyme in 125mM ches buffer, pH 9.6, poised at redox potential of -293mV.

D : C-A spectrum

E : simulation of D

3 unpaired electrons ?

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Electron spin Electron spin –– Electron spin InteractionElectron spin InteractionWhen there is more than one unpaired electron (S>1/2), the interaction between the spins must be considered. This term can be very large. The Hamiltonian for a system with a spin > 1/2 is: H = D [Sz

2 - 1/3 S(S+1) + E/D (Sx2 - Sy

2)] + goβS H

The new terms are D and E/D. D is called the zero-field splitting (ZFS) parameter; E/D is the rhombicity (the ratio between D, the axial splitting parameter, and E, therhombic splitting parameter, at zero field). The minimum value of E/D is 0 for an axial system. The maximum value is 1/2 for a rhombic system. The strength of the ZFS is determined by the nature of the ligands.

So for a completely axial system (E/D = 0), H = D [Sz2 - 1/3 S(S+1)] + goβS H

Consider a case where S = 3/2, i.e., 4 unpaired electrons. These spins can interact to give a total spin moment, referred to as a system spin. There will be four sublevels for ms, where Sz = -3/2, -1/2, 1/2, and 3/2.

The energy for the + or -3/2 level will be: D[9/4-1/3(3/2*5/2)]= D[9/4-5/4]= DThe energy for the + or - 1/2 level will be: -D.

Electron spin Electron spin –– Electron spin InteractionElectron spin Interaction

Magnetic Field

2D

4D

6D

8D

±1/2

±3/2

±5/2

±7/2

±9/2

Ms

g = 18, 0, 0

g = 14, 0, 0

g = 10, 0, 0

g = 6, 0, 0

g = 2, 2, 2g = 2, 4, 4 g = 2, 6, 6 g = 2, 8, 8

g = 2, 10, 10

S = 3/2

S = 5/2

S = 7/2

S = 9/2

D >> hνThe magnitude of the ZFS can be determined by EPR. The populations of each of the doublet has a Boltzmanndistribution. By lowering the temperature to the same energy range as the ZFS and by measuring the EPR amplitude of each doublet, a value of the ZFS can be obtained.

Or we can measure ∆Ms = ±1 transitions, such as +5/2 ↔ +3/2, at higher fields.

Half integer spin and axial ZFS symmetry

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Bull. Kor. Chem. Soc. 27, 193 (2006)

Inorg. Chim. Acta 241, 5 (1996)

Inorg. Chem. 23, 2725 (1984)

EPR of EPR of Fe(III)Fe(III) (S = 5/2, d(S = 5/2, d55))

Magnetic Field

2D

4D

6D

8D

±1/2

±3/2

±5/2

±7/2

±9/2

Ms

g = 18, 0, 0

g = 14, 0, 0

g = 10, 0, 0

g = 6, 0, 0

g = 2, 2, 2g = 2, 4, 4 g = 2, 6, 6 g = 2, 8, 8

g = 2, 10, 10

S = 3/2

S = 5/2

S = 7/2

S = 9/2

D >> hνThe magnitude of the ZFS can be determined by EPR. The populations of each of the doublet has a Boltzmanndistribution. By lowering the temperature to the same energy range as the ZFS and by measuring the EPR amplitude of each doublet, a value of the ZFS can be obtained.

Or we can measure ∆Ms = ±1 transitions, such as +5/2 ↔ +3/2, at higher fields.


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Journal of Molecular Catalysis A : Chemical 116, 405 (1997)

Magnetic Field

2D

4D

6D

8D

±1/2

±3/2

±5/2

±7/2

±9/2

Ms

g = 18, 0, 0

g = 14, 0, 0

g = 10, 0, 0

g = 6, 0, 0

g = 2, 2, 2



Magnetic Field

2D

4D

6D

8D

±1/2

±3/2

±5/2

±7/2

±9/2

Ms

g = 18, 0, 0

g = 14, 0, 0

g = 10, 0, 0

g = 6, 0, 0

g = 2, 2, 2


big E/D

g2z = 4.53, g2y = 4.03, g2x = 4.24

J. Chem. Soc., Dalton Trans., 3001 (2002)

Inorga. Chim. Acta 360, 2944 (2007)

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Coordination Chemistry Reviews 250 (2006) 2271–-2294


Inorganica Chimica Acta 343, 18 (2003)

Inorg. Chem. 40, 4494-4499 (2001)

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EPR of EPR of Mn(II)Mn(II) (S = 5/2, d(S = 5/2, d55))

M eOH

M eOH

DMSO

DMF

DMF

DMF

DMSO

0 2000 4000 6000 8000

Mn4

Mn5

Mn6

Mn7

Field Strength (G)

4

5

6

7


M eOH

M eOH

DMSO

DMF

DMF

DMF

DMSO

0 2000 4000 6000 8000

Mn4

Mn5

Mn6

Mn7

Field Strength (G)

Magnetic Field

2D

4D

6D

8D

±1/2

±3/2

±5/2

±7/2

±9/2

Ms

g = 18, 0, 0

g = 14, 0, 0

g = 10, 0, 0

g = 6, 0, 0

g = 2, 2, 2


55Mn (100%), I=5/2

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Fig. 1 EPR spectra of [Mn(L1)2] in (a) 11 dichloromethane□toluene solution at 300 K, (b) frozen 11 dichloromethane□toluene solution at 77 K, showing computed splittings of t2 orbitals. DPPH = Diphenylpicrylhydrazyl.

J. Chem. Soc., Dalton Trans.1703–1708 (2000)

EPR of EPR of V(IV)V(IV) (S = 1/2, d(S = 1/2, d11))

xx

x

2500 3000 3500 4000

x

Field Strength (G)

(a)

(b)

(c)

(d)

72

52

32

72

52

72

52

32

12

32

72

m =I

72

52

72

52

m =I72

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Dalton trans. 797-803 (2005)

51V (99.8 %), I = 7/2small g anisotroy, big nuclear hyperfine

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EPR of EPR of FeMoFeMo--co co (S = 3/2, 1/2, d(S = 3/2, 1/2, d4343) )

4.33

3.77 2.01

6 7 8 9 10 11 12 13 14Field Strength (kG)

2.09

1.97

1.93

2.172.06

Resting (S=3/2)

lo-CO (S=1/2)

hi-CO (S=1/2)

13CO(I=1/2)

Mo Fe S O C

ENDOR/ ESEEM

EPR of EPR of FeSFeS--clustersclusters

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Interactions measured by EPRInteractions measured by EPR

H = βS.g.H + S.A.I + D[Sz2-1/3 S(S+1) + E/D(Sx

2-Sy2)]

Hyperfine and superhyperfine interactions (electron spin-nuclear spin interaction)

Electron Zeeman interaction (interaction of the spin with the applied field)Spin orbit coupling

Spin-spin interaction

• High sensitivity (<1 µM to 0.1 mM)• No background

• Definitive and Quantitative

* Nuclear quadrupole interation can also be detacted.

ApplicationsApplications

노화 현상

항산화 작용

효소 반응 및 구조

조영제

생분자의 운동

생체내 NO

광합성

생물, 의학 연구

발암 기작

EPR

레이저 재료

고온 초전도체

C60

자성체

광섬유 특성

유기 전도체

반도체 특성재료 연구

고분자 특성

EPR

레이저 재료

고온 초전도체

C60

자성체

광섬유 특성

유기 전도체

반도체 특성재료 연구

고분자 특성

EPR

결정의 결점

자화도 측정

전이 금속, 란탄족, 악틴족 이온

전•반도체의 전도 전자

분자의 여기 상태

물리 연구

EPR

광학 특성

결정장

결정의 결점

자화도 측정

전이 금속, 란탄족, 악틴족 이온

전•반도체의 전도 전자

분자의 여기 상태

물리 연구

EPR

광학 특성

결정장

스핀 트랩

라디칼 반응의 속도론

고분자 반응

산화-환원 반응

촉매

유기금속화학 연구

금속착물

EPR

스핀 트랩

라디칼 반응의 속도론

고분자 반응

산화-환원 반응

촉매

유기금속화학 연구

금속착물

EPR

생물체의 방사선 영향

고고학

방사선 연구 식품의 방사선 효과

EPR생물체의 방사선 영향

고고학

방사선 연구 식품의 방사선 효과

EPR

There are spins everywhere.

25

ReferencesReferences

Good start: John A. Weil, James R. Bolton, John E. Wertz, Electron paramagnetic resonance, John Wiley and Sons, Inc, 1994.

Pulsed EPR: Arthur Schweiger, Gunnar Jeschke, Principles of pulse electron paramagnetic resonance, Oxford University Press, 2001.

Bit old but still good reference: A. Abragam, B. Bleaney, Electron paramagnetic resonance of transition ions, Dover publications, Inc, 1970.

In hurry ? : Russell S. Drago, Physical methods for chemists, Chapters 9, 13, Saunders College Publishing, 1992.

Spin trapping: Rosen, G. M., Britigan, B. E., Halpern, H. J., Pou, S. Free Radicals: Biology and Detection by Spin Trapping. Oxford University Press, 1999

EPR Imaging: Eaton, G. R., Eaton, S. S., Ohno, K. EPR imaging and in vivo EPR: CRC Press, Inc, 1991.

ReferencesReferences

Good start: John A. Weil, James R. Bolton, John E. Wertz, Electron paramagnetic resonance, John Wiley and Sons, Inc, 1994.

Pulsed EPR: Arthur Schweiger, Gunnar Jeschke, Principles of pulse electron paramagnetic resonance, Oxford University Press, 2001.

Bit old but still good reference: A. Abragam, B. Bleaney, Electron paramagnetic resonance of transition ions, Dover publications, Inc, 1970.

In hurry ? : Russell S. Drago, Physical methods for chemists, Chapters 9, 13, Saunders College Publishing, 1992.

Spin trapping: Rosen, G. M., Britigan, B. E., Halpern, H. J., Pou, S. Free Radicals: Biology and Detection by Spin Trapping. Oxford University Press, 1999

EPR Imaging: Eaton, G. R., Eaton, S. S., Ohno, K. EPR imaging and in vivo EPR: CRC Press, Inc, 1991.

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